There are available to the clinical analyst various test devices and methods for the rapid determination of certain constituents in blood samples which if found to be outside the range of a predetermined concentration indicate a potential pathological condition.
In the analysis of blood for analytes such as glucose, lactate, uric acid and ethanol it is common practice to first of all reduce the whole blood sample being tested to plasma by precipitating red blood cells or to blood serum by causing the blood to coagulate. In either case, in the absence of extreme care in preparing the sample, a portion of the red blood cells can be lysed thereby releasing biological impurities such as hemoglobin and bilirubin derived from hemoglobin into the blood sample.
In the analysis of the blood sample for a predetermined analyte, there is typically employed an oxidative reagent system in which an oxidase enzyme selected in accordance with a predetermined analyte such as glucose reacts with the analyte in the presence of oxygen to provide hydrogen peroxide which, in the presence of a peroxidative substance, will oxidize a suitable redox indicator to convert it to its oxidized, colored form and, by visually or instrumentally determining the intensity of the color one can quantitatively determine the concentration of analyte in the blood sample.
In addition to the oxidative system described above, the present invention can be used in combination with reductive system which is represented by the following equations in which glucose is the analyte of interest, ATP represents adenosine triphosphate, ADP represents adenosine diphosphate, AND represents nicotinamide-adenine dinucleotide, NADH represents the reduced form of AND, and NBT represents nitroblue tetrazolium: ##STR1## The intensity of the formazan color produced by this system is directly proportional to the glucose concentration in the system.
In order for such a test to have the required sensitivity, it is essential that the blood specimen being tested be as nearly colorless as is practical under the test conditions in order to avoid interference with the determination of the intensity of the color formed. Accordingly, lysis of the red blood cells and the consequent release of biological substances such as hemoglobin and bilirubin is problematical in the context of these colorimetric determinations due to their interference with the reading of the specimen's reflectance by spectrophotometric means.
In their article appearing in Cancer Res., 47(14), 3624-6 (1987) Anderson et al report that human serum alpha-fetoprotein and albumin were chromatographed on immobilized iminodiacetic acid charged with either Co.sup.+2, Ni.sup.+2, Cu.sup.+2 or Zn.sup.+2. Both proteins were bound to Cu.sup.+2 and were partially resolved by affinity elution with imidazole.
Berk et al report in Biomed. Appln. Immobilized Enzymes & Proteins, Vol. 1, Pp. 297-317 that bilirubin can be removed from blood by affinity-competition chromatography over an albumin-agarose gel.
It is an object of the present invention to provide a novel method for the removal of extraneous biological substances such as hemoglobin and/or bilirubin from samples of blood serum or plasma.
It is a further object to provide such a method which renders the blood sample more amenable to the colormetric analysis of various analytes contained therein.